The broad goal of this project is to learn more about how intercellular relationships modulate the development of neuronal phenotypes. Specifically, the hypothesis will be tested that the various subsets of spinal cholinergic neurons develop their characteristic differences in response to cues encountered on different migratory pathways. The following specific aims are designed to begin a detailed examination of this hypothesis: 1) establishment of the subset identities of cholinergic neurons in pre- and early migratory stages of development. Cholinergic neurons are identified by immunocytochemical detection of choline acetyltransferase (ChAT) or by in situ hybridization of probes for ChAT messenger RNA. Tritiated thymidine autoradiography is combined with one of the above techniques to aid in the determination of migration patterns of cholinergic neurons; 2) identification and characterization of the migration routes used by spinal cholinergic neurons. The approach for this aim is to analyze immunocytochemical preparations to determine the developmental relationships between ChAT-positive cells and potential migratory pathways provided by radial glia and axons of the commissural pathway. Immunocytochemistry will also be used to determine if these potential migratory routes are associated with different cell specific molecules; and 3) development of a topologically accurate in vitro preparation of embryonic rat spinal cord. This preparation is important for experiments designed to identify the exact sites of origin of cholinergic spinal neurons, and to determine whether different migratory routes are causally related to the development of diverse phenotypes by these cells, Thus, a primary emphasis of this project is the epigenetic modulation of neuronal differentiation; a critical concern for understanding the generation of neuronal diversity.